Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause inflammation, fluid secretion, and cell death. Using a gene trap insertional mutagenesis screen in the human colonic epithelial cell line Caco-2, we identified 45 host genes necessary for TcdB-mediated cytotoxicity. We performed detailed follow up on one of these hits, Poliovirus Receptor Like-3 (PVRL3). Disruption of PVRL3 protein expression using gene-trap mutagenesis, shRNA, or CRISPR-Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap and CRISPR mutants resulted in full restoration of TcdB-mediated cell death. Purified receptor ectodomain bound TcdB directly by pull-down outside of the CROPS domain, which had previously been hypothesized to be the receptor binding domain. Pretreatment of cells with a monoclonal antibody specific for PVRL3 or pre-binding TcdB with purified receptor ectodomain also inhibited cytotoxicity. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest that PVRL3 can serve as a new target for the prevention of TcdB-induced necrosis in C. difficile infection.